Agent for accelerating growth of pluripotent stem cells

ABSTRACT

An object of the present invention is to provide a material capable of further accelerating growth of pluripotent stem cells, such as pluripotent stem cells, without impairing pluripotency thereof. In other words, the invention is an agent for accelerating growth of pluripotent stem cells, containing a β-nicotinamide mononucleotide or a pharmaceutically acceptable salt thereof, and a solvate thereof as an active ingredient; and is a method for culturing pluripotent stem cells, including culturing pluripotent stem cells in a culture medium that contains a β-nicotinamide mononucleotide or a pharmaceutically acceptable salt thereof, and a solvate thereof.

TECHNICAL FIELD

The present invention relates to a material capable of furtheraccelerating growth of pluripotent stem cells without impairingpluripotent differentiation potential thereof, and a method forculturing pluripotent stern cells in which the material is used.

Priority is claimed on Japanese Patent Application No. 2017-015313,filed Jan. 31, 2017, the content of which is incorporated herein byreference.

BACKGROUND

Pluripotent stem cells are undifferentiated cells having a self-renewalability and are cells capable of pluripotent differentiating intovarious cells. In recent years, regenerative medicine in whichpluripotent stern cells and cells differentiation-induced from thepluripotent stem cells are transplanted into damaged tissues of apatient to regenerate their functions has been actively studied. Inregenerative medicine, because large amounts of pluripotent stern cellsand differentiated cells thereof are required to be prepared,development of methods for efficiently growing pluripotent stem cells isalso active. In particular, pluripotent stern cells lose theirpluripotency during culture in many cases, and thus a method for growingpluripotent stem cells while maintaining their pluripotency has beenrequired.

As a method for culturing pluripotent stem cells, for example, it isreported that mesenchymal stem cells efficiently grow by culturing themin a medium containing nicotinamide (NAM) and a fibroblast growth factor4 (FGF4) (refer to, for example, Patent Literature 1). In addition, itis also reported that NAM resolves loss of pluripotency of pluripotentstem cells and impairment of reprogramming (refer to, for example,Non-Patent Literature 1). In addition, it is reported that, when inducedpluripotent stem cells (iPS cells) are cultured in the presence of NAM,NAM suppresses the function of sirtuins or PARP, and thereby iPS cellsof which gene expression patterns are similar to those of embryonic stemcells (ES cells) can be efficiently manufactured (refer to, for example,Patent Literature 2).

Meanwhile, nicotinamide mononucleotide (NMN) is a biosyntheticintermediate metabolite of the coenzyme NAD⁺. In recent years, it hasbeen reported that NMN exhibits an effect of ameliorating insulinsecretory ability in senescent mice, exhibits an effect of drasticallyameliorating insulin sensitivity and secretion in a mouse model withtype 2 diabetes caused by high-fat diet and aging (refer to, forexample, Patent Literature 3), and exhibits an effect of significantlyenhancing a mitochondrial function of aged muscle. In addition, it hasbeen reported that administration of NMN is useful for ameliorating orpreventing symptoms of various age-related diseases such as obesity,elevated blood triglyceride and low-density lipoprotein cholesterollevels, decreased insulin sensitivity, decreased memory ability, andocular function deterioration such as macular degeneration (refer to,for example, Patent Literature 4).

CITATION LIST Patent Literature

-   [Patent Literature 1]

Published Japanese Translation No. 2015-507921 of the PCT InternationalPublication

-   [Patent Literature 2]

PCT International Publication No. WO2011/102333

-   [Patent Literature 3]

U.S. Pat. No. 7737158

-   [Patent Literature 4]

PCT International Publication No. WO2014/146044

Non-Patent Literature

-   [Non-Patent Literature 1]

Son, et al., STEM CELLS, 2013, vol. 31, p. 1121-1135.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a material capable offurther accelerating growth of pluripotent stem cells without impairingpluripotency thereof, and a method for culturing pluripotent stern cellsin which the material is used.

Solution to Problem

As a result of intensive studies to achieve the above-described object,the inventors of the present invention have found that growth ofpluripotent stem cells is accelerated in the presence of β-nicotinamidemononucleotide (β-NMN) and pluripotency thereof is not impaired, andtherefore have completed the invention.

In other words, the invention provides the following agent foraccelerating growth of pluripotent stem cells, a method for culturingpluripotent stern cells, and a method for accelerating growth ofpluripotent stern cells.

-   [1] An agent for accelerating growth of pluripotent stem cells,    containing, as an active ingredient: a β-nicotinamide mononucleotide    or pharmaceutically acceptable salt thereof; and a solvate thereof.-   [2] The agent according to [1], which is added at 0.01 to 5 mM into    a culture medium for pluripotent stem cells in terms of    β-nicotinamide mononucleotide.-   [3] The agent according to [1] or [2], which is used for    accelerating growth of one or more kinds of pluripotent stem cells    selected from the group consisting of embryonic stern cells, induced    pluripotent stern cells, and mesenchymal stem cells.-   [4] A method for culturing pluripotent stem cells, including    culturing pluripotent stem cells in a culture medium that contains a    β-nicotinamide mononucleotide or a pharmaceutically acceptable salt    thereof, and a solvate thereof.-   [5] The method according to [4], in which a concentration of the    β-nicotinamide mononucleotide of the culture medium is 0.01 to 5 mM.-   [6] The method according to [4] or [5], in which the pluripotent    stem cells are one or more kinds selected from the group consisting    of embryonic stem cells, induced pluripotent stem cells, and    mesenchymal stem cells.-   [7] A method for accelerating growth of pluripotent stem cells,    including culturing pluripotent stem cells in a culture medium that    contains a β-nicotinamide mononucleotide or a pharmaceutically    acceptable salt thereof, and a solvate thereof.

Advantageous Effects of Invention

The agent for accelerating growth of pluripotent stem cells according tothe present invention acts on pluripotent stem cells such as inducedpluripotent stem cells (iPS cells), embryonic stem cells (ES cells) andthe like thereby can accelerate growth thereof while maintaining theirpluripotency. For this reason, by incorporating the agent foraccelerating growth of pluripotent stem cells in a culture medium,larger amounts of pluripotent stern cells can be efficiently prepared.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing results of measuring absorbance values at 450nm (ref. 595 nm) for each β-NMN concentration of a culture medium withWST assay on wells in which iPS cells (201B7) are cultured in theculture medium to which β-NMN is added in Example 1.

FIG. 2 is a graph showing results of measuring absorbance values at 450nm (ref. 595 nm) for each of β-NMN concentration or NAM concentration ofa culture medium with WST assay on wells in which iPS cells (201B7) arecultured in the culture medium to which β-NMN or NAM is added in Example2.

4 FIG. 3 is a graph showing results of measuring absorbance values at450 nm (ref. 595 nm) for each of β-NMN concentration or NAMconcentration of a culture medium with WST assay on wells in which iPScells (253G1) are cultured in the culture medium to which β-NMN or NAMis added in Example 3.

FIG. 4 is a graph showing results of measuring absorbance values at 450nm (ref. 595 nm) for each β-NMN concentration of a culture medium withWST assay on wells in which iPS cells (201B7) are cultured in theculture medium to which both ROCK inhibitor and β-NMN are added inExample 4.

FIG. 5 is a graph showing results of measuring absorbance values at 405nm for each βNMN concentration of a culture medium by performing analkaline phosphatase measurement test on wells in which iPS cells(201B7) are cultured in the culture medium to which both ROCK inhibitorand β-NMN are added in Example 4.

FIG. 6 is a graph showing results of flow cytometry of iPS cells (201B7)which are stained with an anti-SSEA4 antibody after being cultured in aβ-NMN-free culture medium in Example 6. FIG. 7 is a graph showingresults of flow cytometry of iPS cells (201B7) which are stained with ananti-SSEA4 antibody after being cultured in a culture medium containing0.25 mM of β-NMN in Example 6.

FIG. 8 is a graph showing results of flow cytometry of iPS cells (201B7)which are stained with an anti-SSEA4 antibody after being cultured in aculture medium containing 1 mM of β-NMN in Example 6.

FIG. 9 is a graph showing results of flow cytometry of iPS cells (201B7)which are stained with an anti-low sulfated keratan sulfate antibody(R10G) after being cultured in a β-NMN-free culture medium in Example 6.

FIG. 10 is a graph showing results of flow cytometry of iPS cells(201B7) which are stained with an anti-low sulfated keratan sulfateantibody (R10G) after being cultured in a culture medium containing 0.25mM of β-NMN in Example 6.

FIG. 11 is a graph showing results of flow cytometry of iPS cells(201B7) which are stained with an anti-low sulfated keratan sulfateantibody (R10G) after being cultured in a culture medium containing 1 mMof β-NMN in Example 6.

DESCRIPTION OF EMBODIMENTS

In the present invention and the specification of the presentapplication, pluripotent stem cells are undifferentiated cells whichhave self-renewal ability and pluripotency (an ability to differentiateinto various cell types), and are preferably pluripotent stein cellsthat can differentiate into any of ectodermal, mesodermal, andendodermal cells. Examples of pluripotent stem cells include ES cells,iPS cells, mesenchymal stem cells, and the like.

An agent for accelerating growth of pluripotent stern cells according tothe present invention (hereinafter referred to as the“growth-accelerating agent of the invention” in some cases) contains NMN(chemical formula: C₁₁H₁₅N₂O₈P) as an active ingredient, and is added toa culture medium when culturing pluripotent stern cells. By culturingpluripotent stem cells in the presence of NMN, pluripotent stem cellscan grow more efficiently while maintaining pluripotency thereof.

Regarding NMN, there are two types of α and β as optical isomers, butNMN, which is used as the active ingredient of the growth-acceleratingagent of the present invention, is β-NMN (CAS number: 1094-61-7). Astructure of β-NMN is shown below.

β-NMN may be prepared by any method. For example, β-NMN obtained bypurifying β-NMN artificially synthesized by a chemical synthesis method,an enzymatic method, a fermentation method, or the like can be used asthe active ingredient. In addition, because β-NMN is a component widelypresent in a living body, β-NMN obtained by extraction and purificationfrom natural raw materials such as animals, plants, and microorganismscan also be used as the active ingredient. Furthermore, commerciallyavailable purified β-NMN may be used.

As a chemical synthesis method for synthesizing β-NMN, for example,β-NMN can be produced by reacting NAM with L-ribose tetraacetate, andphosphorylating the obtained nicotinamide mononucleotide. In addition,as an enzymatic method, for example, β-NMN can be produced from NAM and5′-phosphoribosyl-1′-pyrophosphate (PRPP) by nicotinamidephosphoribosyltransferase (NAMPT). As a fermentation method, forexample, β-NMN can be produced from NAM using a metabolic system of amicroorganism expressing NAMPT.

The active ingredient of the growth-accelerating agent of the inventionmay be pharmaceutically acceptable salts of β-NMN. The pharmaceuticallyacceptable salt of β-NMN may be an inorganic acid salt or an organicacid salt having a basic site such as an amine. Examples of acidsconstituting such acid salts include acetic acid, benzenesulfonic acid,benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid,fumaric acid, gluconic acid, glutamic acid, hydrobromic acid,hydrochloric acid, isethionone acids, lactic acid, maleic acid, malicacid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid,pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuricacid, tartaric acid, p-toluenesulfonic acid, and the like. In addition,the pharmaceutically acceptable salt of β-NMN may be an alkali salt oran organic salt having an acidic site such as a carboxylic acid.Examples of bases constituting such acid salts include bases which arealkali metal salts or alkaline earth metal salts and which are inducedfrom bases such as sodium hydride, potassium hydroxide, calciumhydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide,zinc hydroxide, ammonia, trimethyl ammonia, triethyl ammonia, ethylenediamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine,N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)-aminomethane, and tetramethyl ammonium hydroxide.

The active ingredient of the growth-accelerating agent of the inventionmay be a solvate of free β-NMN or pharmaceutically acceptable saltsthereof. Examples of solvents that form the above-mentioned solvateinclude water, ethanol, and the like.

The growth-accelerating agent of the invention may contain other activeingredients in addition to β-NMN. It is possible to appropriately selectthe other active ingredients from, for example, a component known toenhance survival efficiency or growth efficiency of pluripotent steincells, or a component known to have an effect of maintaining anundifferentiated state of pluripotent stem cells; and to use them.Examples of components that enhance survival efficiency of pluripotentstem cells include Rho kinase (ROCK) inhibitors. In addition, examplesof components for maintaining an undifferentiated state of pluripotentstem cells include FGF-2 and TGFβ superfamily. TGFβ superfamily includesTGF-β, activin, NODAL, and the like. The other active ingredients to beused in combination with β-NMN may be only one kind or a combination oftwo or more kinds thereof.

By incorporating the growth-accelerating agent of the invention in aculture medium when pluripotent stem cells are cultured, growth of thepluripotent stem cells can be accelerated while maintainingdifferentiation potential thereof. An amount of the agent of theinvention to be added to a culture medium is not particularly limited aslong as the amount is an amount in which a concentration is sufficientto accelerate growth of pluripotent stern cells as compared to a case ofculture in a culture medium not containing the agent, and the amount canbe appropriately adjusted in consideration of the type of pluripotentstern cells, the balance with other components of the culture medium,and the like. In a case where a concentration of β-NMN in the culturemedium is too low, a growth-accelerating effect on pluripotent stemcells may be weak, and in a case where β-NMN is excessively contained,growth may be suppressed conversely. A content of thegrowth-accelerating agent of the present invention in a culture mediumis preferably a content in which a β-NMN concentration is 0.01 to 5 mM,more preferably 0.05 to 2 mM, even more preferably 0.1 to 1 mM. When aβ-NMN concentration is within the above range, growth of pluripotentstern cells can be sufficiently accelerated while maintainingpluripotency thereof. A growth-accelerating effect of β-NMN is superiorto other NAM-related substances such as NAM, nicotinic acid, andnicotinamide riboside.

Culturing of pluripotent stem cells in the presence of thegrowth-accelerating agent of the invention can be performed by generalmethods except that a culture medium contains the agent of theinvention. For example, as a culture medium, it is possible to use amedium generally used for maintenance or growth of pluripotent sterncells, and a medium used for culture of animal cells. In addition, it ispossible to use various commercially available culture media forpluripotent stem cells. In the present invention, examples of media thatcontain the agent of the invention and are used for culture ofpluripotent stem cells include Eagle's Minimal Essential Medium (MEM),Dulbecco's modified Eagle's medium (DMEM), Minimum Essential MediumEagle-α (αMEM), Iscove's Modified Dulbecco's Medium (IMDM), F-12 medium,F-10 medium, DMEM/F12 medium, RPMI-1640 medium, mesenchymal cell basalmedium (MSCBM), E8 (Essential 8) medium, TeSR-E8 medium, mTeSR1 medium,and the like. If necessary, amino acids, inorganic salts, vitamins,antibiotics, and the like may be added to these media.

In these culture media, a component known to enhance survival efficiencyor growth efficiency of pluripotent stem cells, or a component known tohave an effect of maintaining an undifferentiated state of pluripotentstern cells, and the like may be appropriately contained in addition tothe growth-accelerating agent of the invention. As these components,components described above can be used.

In addition, culture conditions can be set as general culture conditionsfor culturing animal cells, and may be suitably modified as necessary.For example, culture can be performed at a culture temperature of 30 to40° C., a CO₂ concentration of 1 to 10% by volume, and an O₂concentration of 0.1 to 25% by volume.

As pluripotent stem cells in which growth is accelerated by thegrowth-accelerating agent of the invention, pluripotent stern cellsderived from mammal are preferable; pluripotent stern cells derived fromhuman are more preferable; and pluripotent stein cells derived fromhuman are particularly preferable. As pluripotent stem cells in whichgrowth is accelerated by the agent of the invention, ES cells, iPScells, or mesenchymal stem cells are preferable; ES cells, iPS cells, ormesenchymal stem cells which are derived from human are more preferable;and ES cells or iPS cells which are derived from human are even morepreferable.

EXAMPLES

Next, the present invention will be described in more detail by showingexamples, but the invention is not limited to the following examples.

Example 1

iPS cells were cultured in a culture medium containing β-NMN, and aneffect of β-NMN on growth was examined.

As iPS cells, 201B7 line which is human iPS cells was used. For iPScells, an E8 medium (LTC) containing 19.4 mg/L of insulin, 10.7 mg/L oftransferrin, 100 μg/L of bFGF, 2 μg/L of TGFβ, 14 μg/L of sodiumselenite, 64 mg/L of ascorbic acid, and 543 mg/L of NaHCO₃ in DMEM/F12was used as the basal medium.

First, iPS cells cultured in the basal medium were separated from aculture vessel by a cell-peeling solution and recovered. The recoveredcells were counted and seeded in each well of a Matrigel-coated 96-wellplate at 1000 to 2000 cells/well. At this time, a ROCK inhibitor wasadded to each well. The 96-well plate was cultured at 37° C. for 1 dayto allow the cells to adhere, and then the medium containing the ROCKinhibitor was removed from each well, the medium was replaced with amedium obtained by adding, to the basal medium, β-NMN (Oriental YeastCo., Ltd.) so that a final concentration became 0 to 2 mM, and culturingwas performed for 3 to 4 days.

Thereafter, a growth ability of cells surviving in each well wasmeasured by water-soluble tetrazolium salts (WST) assay. Specifically,after adding WST-1 (manufactured by Nacalai Tesque) to each well andincubating at 37° C. for 1 to 4 hours, absorbance values at 450 nm (ref.595 nm) were measured using a microplate reader (manufactured by BMGLABTECH). The term “absorbance value (450 to 595 nm)” is an absorbancevalue at 450 nm using an absorbance value at 595 nm as a referencevalue, and is specifically a value obtained by subtracting an absorbancevalue at 595 nm from an absorbance value at 450 nm.

The results of the absorbance values at 450 nm (ref. 595 nm) for eachβ-NMN concentration of the medium are shown in FIG. 1 . The absorbancevalues at 450 nm (ref. 595 nm) of the cells cultured in the mediumcontaining 0.1 mM or more of β-NMN was greater than that of the cellscultured in the β-NMN-free medium (β-NMN: 0 mM), and therefore it wasfound that growth of iPS cells was accelerated in the presence of β-NMN.In particular, when a concentration of β-NMN was 0.1 to 0.2 mM, theabsorbance value increased in a concentration-dependent manner; when aconcentration of β-NMN was 0.2 to 0.6 mM, the absorbance value was thesame; and when a concentration of β-NMN was 0.8 mM or more, theabsorbance value tended to slightly decrease in a β-NMNconcentration-dependent manner.

Example 2

iPS cells were cultured in a culture medium containing β-NMN or NAM, andthe effects of β-NMN and NAM on growth were compared. As iPS cells,201B7 line was used.

Specifically, iPS cells were cultured and the WST assay was performed inthe same manner as in Example 1 except that the medium added afterremoving the medium containing the ROCK inhibitor was changed to aculture medium to which β-NMN was added so that a final concentrationbecame 0, 0.2, or 0.4 mM, or a culture medium to which NAM was added sothat a final concentration became 0, 0.2, or 0.4 mM.

The results of the absorbance values at 450 nm (ref. 595 nm) for eachculture medium are shown in FIG. 2 . In FIG. 2 , “E8+NMN” is the resultsof the medium to which each concentration of β-NMN was added, and“E8+NAM” is the results of the medium to which each concentration of NAMwas added. As shown in FIG. 2 , both the cells cultured in the medium towhich β-NMN was added and the cells cultured in the medium to which NAMwas added have higher absorbance values than cells cultured in the basalmedium, which means that both β-NMN and NAM had a growth-acceleratingeffect on iPS cells. In both the case in which a final concentration ofthe medium was 0.2 mM and the case in which a final concentration of themedium was 0.4 mM, the absorbance value of the cells cultured in theβ-NMN-added medium was significantly higher than that of the cellscultured in the NAM-added medium, and therefore β-NMN was confirmed tohave a higher growth-accelerating effect than NAM.

Example 3

iPS cells were cultured in culture medium containing β-NMN or NAM, andthe effects of β-NMN and NAM on growth were compared. As iPS cells,253G1 line was used.

Specifically, iPS cells were cultured and WST assay was performed in thesame manner as in Example 1 except that 253G1 line was used instead of201B7 line.

The results of the absorbance values at 450 nm (ref. 595 nm) for eachculture medium are shown in FIG. 3 . In FIG. 3 , “E8+NMN” is the resultsof the medium to which each concentration of β-NMN was added, and“E8+NAM” is the results of the medium to which each concentration of NAMwas added. 0 mM on the graph is the result with the E8 medium. As shownin FIG. 3 , the cells cultured in the medium to which β-NMN was addedhave a higher absorbance value than that of the cell cultured in thebasal medium, and β-NMN also has a growth-accelerating effect on the253G1 line. On the other hand, the cells cultured in the medium to whichNAM was added have a slightly higher absorbance value than that of thecells cultured in the basal medium, but no clear growth-acceleratingeffect as in the case of β-NMN could be confirmed. Therefore, it wasfound that a growth-accelerating effect of NAM could not be obtainedsufficiently depending on lines. In addition, as in the results ofExample 2, in both the case in which a final concentration of the mediumwas 0.2 mM and the case in which a final concentration of the medium was0.4 mM, the absorbance value of the cells cultured in the β-NMN-addedmedium was significantly higher than that of the cells cultured in theNAM-added medium, and therefore β-NMN was confirmed to also have ahigher growth-accelerating effect on 253G1 line than NAM. Based on theseresults, β-NMN clearly has a significantly superior growth-acceleratingeffect on various iPS cells than NAM.

Example 4

iPS cells were cultured in a culture medium containing β-NMN, and theeffects of β-NMN on differentiation potential and growth were examined.As iPS cells, a 201B7 line was used. The differentiation potential wasexamined using the enzyme activity of alkaline phosphatase, which is anundifferentiated marker, as an indicator.

<Effect on Growth>

The iPS cells cultured in the basal medium used in Example 1 wereseparated from a culture vessel by a cell-peeling solution andrecovered. The recovered cells were counted and seeded in each well of aMatrigel-coated 96-well plate at 1000 to 2000 cells/well. At this time,a ROCK inhibitor and β-NMN having a final concentration of 0 to 1 mMwere added to each well. The cells in 96-well plate was cultured at 37°C. for 1 day to allow the cells to adhere, and then the mediumcontaining the ROCK inhibitor was removed from each well, the medium wasreplaced with a culture medium obtained by adding, to the basal medium,β-NMN so that a final concentration became 0 to 1 mM, and culturing wasperformed for 3 to 4 days.

Thereafter, WST assay was performed in the same manner as in Example 1.The results of measuring absorbance values at 450 nm (ref. 595 nm) ofeach cells cultured in a medium to which both ROCK inhibitor and β-NMNwere added are shown in FIG. 4 . As shown in FIG. 4 , agrowth-accelerating effect on iPS cells was obtained in a β-NMNconcentration-dependent manner regardless of addition timings of β-NMN.

<Alkali Phosphatase Measurement Test>

First, the iPS cells cultured in the basal medium used in Example 1 wereseparated from a culture vessel by a cell-peeling solution andrecovered. The recovered cells were counted and seeded in each well of aMatrigel-coated 96-well plate at 2000 cells/well. At this time, the ROCKinhibitor and β-NMN having a final concentration of 0 to 1 mM were addedto each well. The cells in 96-well plate was cultured at 37° C. for 1day to allow the cells to adhere, and then the medium containing theROCK inhibitor was removed from each well, the medium was replaced witha medium obtained by adding, to the basal medium, β-NMN so that a finalconcentration became 0 to 1 mM, and culturing was performed for 3 to 4days. During this period, the medium was replaced daily.

Next, after removing the medium in each well, an ethanol-acetonefixation solution was added to fix the cells in each well. After dryingthe wells, a bicarbonate buffer containing p-nitrophenol triphosphateacid, which is an alkaline phosphatase measurement reagent, was addedand incubated at 37° C. for 30 minutes, and then absorbance at 405 nmwas measured using a microplate reader.

The results of the absorbance values at 405 nm of each cells cultured ina medium to which both ROCK inhibitor and β-NMN were added are shown inFIG. 5 . As shown in FIG. 5 , regardless of addition timings of β-NMN,an absorbance value at 405 nm of the cells cultured in the medium towhich β-NMN was added was higher than that of the cells cultured in theβ-NMN-free medium. Therefore, iPS cells were confirmed to be able togrow while maintaining undifferentiation properties.

Example 5

Human mesenchymal stern cells (human MSCs) were cultured in a culturemedium containing β-NMN, and an effect of β-NMN on proliferativeactivity was examined.

Human MSCs purchased from Lonza were used. As a basal medium for humanMSCs, a dedicated maintenance medium purchased from Lonza was used.

Human MSCs were seeded in 96-well plates (Nunc) at 2×10³ cells/well (n=3to 4). They were cultured at 37° C. in a CO₂ incubator to allow them toadhere, and then β-NMN (manufactured by Oriental Yeast Co., Ltd.) wasadded so that a final concentration became 1.0, 0.1, 0.01, or 0.001 mM,and culture was further performed at 37° C. in a CO₂ incubator for 72hours.

Thereafter, proliferative activity of cells in each well was measuredwith WST assay. Specifically, after adding a viable cell count reagentSF (Nacalai Tesque) to each well and incubating at 37° C. for 1 to 4hours, absorbance at 450 nm was measured with the reference wavelengthat 620 nm using a microplate reader (absorbance values at 450 nm (ref.620 nm) (BMG LABTECH). The term “absorbance values at 450 nm (ref. 620nm)” is an absorbance value at 450 nm using an absorbance value at 620nm as a reference value, and is specifically a value obtained bysubtracting an absorbance value at 620 nm from an absorbance value at450 nm. [0045]

The results of the absorbance at 450 nm (ref. 620 nm) in each well foreach β-NMN concentration were shown in Table 1. The addition of β-NMNincreased the absorbance value and accelerated proliferative activity ofhuman MSCs as well as iPS cells.

TABLE 1 β-NMN concentration Absorbance value at450 [mM] nm (ref. 620 nm)0 (not added) 0.569 ± 0.0126 0.001 0.573 ± 0.0312 0.01 0.604 ± 0.05870.1 0.634 ± 0.0411 1 0.615 ± 0.0340

Example 6

The differentiation potential of iPS cells maintained and grown in thepresence of β-NMN was confirmed. As iPS cells, a 201B7 line was used.

Specifically, iPS cells were seeded in a 35 mm dish coated with Matrigel(Corning) at 1.5×10⁴ cells/dish. As culture medium in which β-NMN wasadded to a basal medium (E8 medium) of iPS cells so that a finalconcentration became 0, 0.25, or 1 mM was used. At the time of seeding,Rho-dependent apoptosis was suppressed using a culture medium to which aRock inhibitor was added so that a final concentration became 10 μM. Themedium was replaced daily and the cells were passaged on day 5 and 6.iPS cells cultured for 5 or more passages in the medium to which β-NMNwas added were stained with each of antibodies against SSEA4 and againstlow sulfated keratan sulfate which are undifferentiated markers, andcells expressing the undifferentiated markers were analyzed with a flowcytometer.

FIGS. 6 to 8 are graphs each showing results of flow cytometry of iPScells stained with an anti-SSEA4 antibody after being cultured at β-NMNconcentrations of 0, 0.25, and 1 mM, respectively. FIGS. 9 to 11 aregraphs each showing results of flow cytometry of iPS cells stained withan anti-low sulfated keratan sulfate antibody (R10G) after beingcultured at β-NMN concentrations of 0, 0.25, and 1 mM, respectively.Expression of SSEA4 and low sulfated keratan sulfate was maintained inthe iPS cells cultured in the presence of 0.25 mM or 1 mM of β-NMN as inthe iPS cells cultured in the absence of β-NMN (0 mM of β-NMNconcentration). Based on these results, it was confirmed that cellgrowth can be accelerated while maintaining differentiation potential byculturing iPS cells in the presence of β-NMN.

1-7. (canceled)
 8. A method for accelerating growth of a mesenchymalstem cell, comprising: culturing the mesenchymal stem cell in a culturemedium that comprises a β-nicotinamide mononucleotide, or apharmaceutically acceptable salt thereof, or a solvate thereof, whereina concentration of the β-nicotinamide mononucleotide of the culturemedium is in a range from 0.05 to 2.0 mM.
 9. The method for acceleratinggrowth of a mesenchymal stem cell according to claim 8, wherein theconcentration of the β-nicotinamide mononucleotide of the culture mediumis in a range from 0.05 to 1.0 mM.
 10. The method for acceleratinggrowth of a mesenchymal stem cell according to claim 8, wherein theconcentration of the β-nicotinamide mononucleotide of the culture mediumis in a range from 0.1 to 2.0 mM.
 11. The method for accelerating growthof a mesenchymal stem cell according to claim 10, wherein theconcentration of the β-nicotinamide mononucleotide of the culture mediumis in a range from 0.1 to 1.0 mM.
 12. A method for culturing apluripotent stem cell, comprising: culturing the pluripotent stem cellin a culture medium that comprises a β-nicotinamide mononucleotide, or apharmaceutically acceptable salt thereof, or a solvate thereof, whereina concentration of the β-nicotinamide mononucleotide of the culturemedium is in a range from 0.1 to 2.0 mM, the pluripotent stem cell is atleast one cell selected from the group consisting of an embryonic stemcell and an induced pluripotent stem cell, and the pluripotent stem cellgrows while maintaining pluripotency thereof.
 13. The method forculturing a pluripotent stem cell according to claim 12, wherein theconcentration of the β-nicotinamide mononucleotide of the culture mediumis in a range from 0.1 to 1.0 mM.
 14. The method for culturing apluripotent stem cell according to claim 12, wherein the concentrationof the β-nicotinamide mononucleotide of the culture medium is in a rangefrom 0.2 to 0.8 mM.